1. Field of the Invention
The present invention relates to novel methods of making a fermented food product, especially fermented dairy products. Further, the invention relates to novel mutant strains of Lactobacillus bulgaricus (L. bulgaricus) and methods of making them which are useful in the preparation of yogurt and other fermented food products. The invention also relates to novel mutant genes and organisms transformed with such genes.
2. Background Art
A variety of food products are available worldwide which depend on active bacterial cultures in the final form of the food product for flavor, preservation of quality, claimed health benefits and/or pH. Examples are fermented vegetable products, such as sauerkraut from cabbage and pickles from cucumbers; fermented fish products such as fish paste or burongdalog; fermented seeds such as coffee or cocoa beans; fermented starch-rich food products; fermented meat products, fermented cassava; or fermented fruit juices. Particularly important are fermented dairy products such as yogurt, sour cream, creme frache; buttermilk, and the like. These fermented dairy products depend, to a certain degree, on organisms which metabolize lactose to produce flavor and lower the pH to preserve the food. These products can be especially important to those people with lactose intolerance; i.e., the inability or difficulty in metabolizing lactose.
Yogurt is an extremely popular fermented dairy product. Microbiologically, yogurt may be defined in the United States and many other countries as the end product of a controlled fermentation of milk with a mixture of Streptoccocus thermophilus (S. thermophilus) and L. bulgaricus. The fermentation is carried out within a temperature range of 35.degree.-45.degree. C. with about 42.degree.-45.degree. C. being preferred. Early in the incubation, S. thermophilus grows rapidly dominating the fermentation and lowering the oxidation-reduction potential of the system. L. bulgaricus grows slowly during the early fermentation but liberates sufficient amounts of peptides and amino acids to stimulate growth of S. thermophilus which results in moderate production of lactic acid, acetic acid, acetaldehyde, diacetyl and formic acid. When the pH of the fermentation mix is sufficiently lowered to around 5.5, the rapid growth of S. thermophilus is arrested and the growth of L. bulgaricus is favored. Depletion of oxygen from the system and the availability of formate is believed to stimulate such growth. The major portion of lactic acid and acetaldehyde necessary for the characteristic flavor of yogurt is contributed by the L. bulgaricus which has been aided by the initial activity of the S. thermophilus component. When the pH drops further to about 5.0 or less the product is cooled to about 10.degree. C. or less for storage. Although the rate of production of lactic acid is diminished under the normal storage conditions of yogurt (4.degree.-10.degree. C.), production of lactic acid continues to such a degree that the yogurt product is, depending on strain or process, rendered unpalatable (sour after 3 or 4 weeks. Attempts to further slow or arrest the production of lactic acid during storage while maintaining viable organisms have been largely unsuccessful. Preservatives have been tried but have the undesirable side effect of affecting the viability or killing the yogurt organism. Attempts at mutating the organism directly; e.g., through chemical or other mutagenesis techniques have not only produced organisms with decreased lactic acid production at lower temperature and pH, but organisms also showing a proportional decrease in lactic acid production at fermentation temperatures and a decreased growth, thus producing an unsatisfactory yogurt product or products which are limited to the strain which has been mutated (with an unknown effect), which can not be easily transferred to new starters (see U.S. Pat. No. 4,734,361 to Meiji Milk Products wherein a method is described for isolating a naturally-occurring variant of Lactobacillus bulgaricus). This temperature-sensitive organism produces less than 0.1% lactic acid at 10.degree. C. for 7 days. This method is dependent on a strain being present which matches the description of the invention, and, to date, only one such natural isolate is known to exist, OLL 1074. Currently no cultures, especially of L. bulgaricus, are available which combine the desired needs of texture and taste while maintaining the extended lowered metabolism under storage temperature, have the site of mutation defined and are transferable to new starters.
It would be useful to construct a L. bulgaricus which exhibited both decreased production of lactic acid at the storage temperature of yogurt yet still retained acceptable levels of activity under production conditions of yogurt where the mutation is defined and which could be transferable to new starters starting from any single strain of L. bulgaricus and remains viable in the yogurt product. It would also be useful to have such organism produce less acid at temperatures below about 20.degree. C. or at about pH 5.5 or lower in order to give more flexibility during production and handling after production. Furthermore, it would aid in the manufacture of fermented products to have the fermentation slow or stop after reaching a certain range of temperature and/or pH rather than using a quick refrigeration to decrease the rate of acid formation.